1. Field of the Invention
The invention pertains to novel methods of separating and purifying plasmid DNA isoforms using ultrafiltration.
2. Description of Related Art
There is considerable interest in using plasmids for gene therapy and DNA-based vaccines. These applications include: cancer (Allovectin-7®—Vical Inc.); cystic fibrosis (PLASmin™—Copernicus Therapeutics); hemophilia (Transkaryotic Therapies Inc.); and muscular dystrophy (MyoDys®—Transgene), among others.
Plasmids are circular, double-stranded extrachromosomal DNA that are produced by many bacteria in a plectonemic supercoiled form, often at high copy numbers. Large-scale production of plasmid DNA is performed in an appropriate bioreactor, with the plasmid released into the fluid medium by controlled cell lysis. One of the challenges in the preparation of a viable therapeutic product is the purification of the plasmid DNA, including removal of host cell proteins, genomic DNA, and RNA as well as the separation of the desired supercoiled isoform from the undesired linear and open-circular (i.e. nicked) isoforms. Current FDA guidelines recommend that at least 90% of the plasmid DNA in the final product must be in the supercoiled form. Therefore, the commercial need to obtain highly purified supercoiled plasmid DNA is extremely high.
For therapeutic applications, there is a particular and critical need for high purity product, which means that the bacterial host cell proteins, genomic DNA fragments, RNA, and endotoxins must all be removed. Moreover, the therapeutic effectiveness is dependent on the specific plasmid DNA morphology (i.e. isoform).
A variety of purification strategies have been examined for the large-scale separation of plasmid DNA including precipitation, aqueous two-phase separation, and also adsorption using monolithic columns. However, the dominant techniques used for the purification of plasmid DNA are chromatographic at this writing. These chromatographic techniques include size exclusion chromatography (SEC), anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), and thiophilic-aromatic chromatography (TAC).
There are a number of challenges associated with the use of chromatographic systems for large-scale DNA purification. In particular, chromatographic processes suffer from many of the following limitations: limited resolution, low binding capacity, mass transfer limitations, and high process cost. For example, although HIC can provide reasonable separation of the open-circular and supercoiled forms, the separation between supercoiled and linear forms has heretofore been poor. Conflicting results have been reported for the use of AEX chromatography for DNA separation. Resolution of plasmid isoforms in SEC has been poor, and large-scale applications of SEC tend to be very time-consuming and expensive. Also, the pores of typical chromatographic resins are similar in size to plasmid DNA. Thus, much of the internal surface area of the resin is inaccessible to the DNA molecules and this in turn greatly increases the resin volume needed to purify the plasmid DNA.
Ultrafiltration is used extensively in bio-processing for the concentration and buffer exchange of therapeutic proteins, both for final product formulation and for pre-conditioning of feed streams prior to chromatography. Several previous studies have examined the potential of using ultrafiltration for the purification of plasmid DNA. Hirasaki et al., J. Membr. Sci., 106: 123-129 (1995), in the only study to date that compared the UF transmission of different plasmid isoforms, found that the transmission of the linear isoform was slightly larger than that for the supercoiled form. However, the resolution was too low for effective separation. Kong et al., J. Membr. Sci., 280: 824-831 (2006) investigated plasmid DNA transmission through microfiltration membranes and observed small differences in the overall rate of plasmid transmission depending upon the relative amount of the different isoforms, but no data were provided for the purification of the desired supercoiled form.
A need therefore remains for a technique for accurately and efficiently purifying specific plasmid DNA isoforms using a separation technique that is applicable for large-scale production of the desired isoform.